Journal: Nature chemical biology

Article Title: Direct stimulation of de novo nucleotide synthesis by O-GlcNAcylation.

doi: 10.1038/s41589-023-01354-x

Figure Lengend Snippet: Fig. 2 | OGT interacts with and O-GlcNAcylates PRPS1. a, Reciprocal endogenous immunoprecipitation (IP) assay between OGT and PRPS1 in HEK293T cells (n = 3). b, GST, GST-OGT and GST-PRPS1 proteins were expressed and purified from E. coli and subjected to in vitro pulldown of endogenous PRPS1 or OGT from HEK293T cells (n = 3). c–f, The indicated siRNAs were introduced into H1299 cells (c); H1299 cells were then cultured with or without glucose for 24 h (d), transfected with or without HA-OGT plasmids (e) and transfected with or without HA-OGA plasmids (f). Cells from all groups were transfected with vector or FLAG-PRPS1 plasmids. IP and immunoblot analyses were conducted with anti-FLAG agarose beads and the indicated antibodies (n = 3). g,h, PRPS1 O-GlcNAcylation analysis using a chemoenzymatic labeling method in HEK293T cells. g, Immunoblot of O-GlcNAcylated PRPS1 in elution and total PRPS1 in input.

Article Snippet: The following primary antibodies were used for immunoblotting: PRPS1 (Proteintech, 15549-1-AP); phospho-PRPS1 (S180) (Thermo Fisher Scientific, PA5-106230); PRPS2 (Novus, NBP1-31435); OGT (Cell Signaling Technology, 24083); OGA (Proteintech, 14711-1-AP); AMPKα (Cell Signaling Technology, 2532); phospho-AMPKα (Thr172) (Cell Signaling Technology, 2535); ACC (Cell Signaling Technology, 3662); phospho-ACC (S79) (Cell Signaling Technology, 3661); β-tubulin (Proteintech, 66240-1-Ig); β-actin (Proteintech, 60008-1-Ig); γ-H2AX (S139) (Cell Signaling Technology, 9718); histone H3 (Millipore, 06-755); O-GlcNAc (RL2) (Abcam, ab2739); O-GlcNAc (CTD110.6) (Cell Signaling Technology, 9875); O-GlcNAc (18B10.C7) (Thermo Fisher Scientific, MA1-038); FLAG (Sigma-Aldrich, F3165); HA (BioLegend, MMS-101P, clone 16B12); HA (Cell Signaling Technology, 3724); and Myc (Santa Cruz Biotechnology, sc-40).

Techniques: Immunoprecipitation, Purification, In Vitro, Cell Culture, Transfection, Plasmid Preparation, Western Blot, Labeling

Journal: Nature chemical biology

Article Title: Direct stimulation of de novo nucleotide synthesis by O-GlcNAcylation.

doi: 10.1038/s41589-023-01354-x

Figure Lengend Snippet: Fig. 4 | PRPS1 O-GlcNAcylation inhibits its phosphorylation by AMPK. a,b, The indicated siRNAs were introduced into H1299 cells (a); the Myc-vector or Myc-OGT plasmids were introduced into H1299 cells (b). Immunoblot analyses were conducted with indicated antibodies (n = 4). c,d, HEK293T cells were transfected by indicated plasmids. Subsequent immunoprecipitation (IP) and immunoblotting procedures were carried out to examine interaction with AMPK (c) (n = 3) and S180 phosphorylation levels (d) (n = 4) of PRPS1 WT, S83A, T166A, 2A or S180A. e,f, The recombinant O-GlcNAcylated and non- O-GlcNAcylated GST-PRPS1 proteins obtained from the in vitro O-GlcNAcylation assays were subjected to in vitro pulldown of AMPK (e) or AMPK in vitro kinase assays (f) (n = 3). g, The stoichiometry analysis of S180 phosphorylation on O- GlcNAcylated or non-O-GlcNAcylated peptides (aa 160–185) by AMPK through MS (n = 3). h, The FLAG-PRPS1 plasmids were introduced into Ampkα1/α2 WT or depleted MEFs. Indicated cells were treated with or without 2 mM AICAR for 1 h. IP and immunoblot analyses were performed with anti-FLAG beads and indicated antibodies (n = 2). i, HEK293T cells were transfected with indicated plasmids. IP and immunoblot analyses were performed with anti-FLAG beads and indicated

Article Snippet: The following primary antibodies were used for immunoblotting: PRPS1 (Proteintech, 15549-1-AP); phospho-PRPS1 (S180) (Thermo Fisher Scientific, PA5-106230); PRPS2 (Novus, NBP1-31435); OGT (Cell Signaling Technology, 24083); OGA (Proteintech, 14711-1-AP); AMPKα (Cell Signaling Technology, 2532); phospho-AMPKα (Thr172) (Cell Signaling Technology, 2535); ACC (Cell Signaling Technology, 3662); phospho-ACC (S79) (Cell Signaling Technology, 3661); β-tubulin (Proteintech, 66240-1-Ig); β-actin (Proteintech, 60008-1-Ig); γ-H2AX (S139) (Cell Signaling Technology, 9718); histone H3 (Millipore, 06-755); O-GlcNAc (RL2) (Abcam, ab2739); O-GlcNAc (CTD110.6) (Cell Signaling Technology, 9875); O-GlcNAc (18B10.C7) (Thermo Fisher Scientific, MA1-038); FLAG (Sigma-Aldrich, F3165); HA (BioLegend, MMS-101P, clone 16B12); HA (Cell Signaling Technology, 3724); and Myc (Santa Cruz Biotechnology, sc-40).

Techniques: Phospho-proteomics, Plasmid Preparation, Western Blot, Transfection, Immunoprecipitation, Recombinant, In Vitro

Journal: Nature chemical biology

Article Title: Direct stimulation of de novo nucleotide synthesis by O-GlcNAcylation.

doi: 10.1038/s41589-023-01354-x

Figure Lengend Snippet: Fig. 5 | PRPS1 O-GlcNAcylation promotes tumor growth. a,b, The indicated plasmids were reintroduced into PRPS1-deleted H1299 cells. Cells were labeled with 10 µM EdU for 30 min, and EdU-positive cells were examined by immunostaining. The quantification is shown in the middle panel. Protein levels were examined by western blot in the right panel (a) (n = 3). Cell growth was analyzed by measuring OD450 in cell viability assays. The box-and-whisker plots show all the points from the minimum to maximum values (b) (n = 5). c,d, H1299 cells were transfected with the indicated constructs and injected subcutaneously into flank regions of nude mice. Mice were treated with PBS or etoposide (20 mg kg−1, twice a week for 3 weeks), and xenograft volumes were measured on the indicated days (c). At the end of the experiment, mice were euthanized, and tumors were weighed. The xenografts were subjected to label-free LC–MS/MS analysis for nucleotide monophosphate levels (d) (n = 5). e, The indicated H1299 cells were seeded and treated with indicated concentrations of etoposide. The colony-forming efficiencies were analyzed (n = 3). f, The tumor weight ratios

Article Snippet: The following primary antibodies were used for immunoblotting: PRPS1 (Proteintech, 15549-1-AP); phospho-PRPS1 (S180) (Thermo Fisher Scientific, PA5-106230); PRPS2 (Novus, NBP1-31435); OGT (Cell Signaling Technology, 24083); OGA (Proteintech, 14711-1-AP); AMPKα (Cell Signaling Technology, 2532); phospho-AMPKα (Thr172) (Cell Signaling Technology, 2535); ACC (Cell Signaling Technology, 3662); phospho-ACC (S79) (Cell Signaling Technology, 3661); β-tubulin (Proteintech, 66240-1-Ig); β-actin (Proteintech, 60008-1-Ig); γ-H2AX (S139) (Cell Signaling Technology, 9718); histone H3 (Millipore, 06-755); O-GlcNAc (RL2) (Abcam, ab2739); O-GlcNAc (CTD110.6) (Cell Signaling Technology, 9875); O-GlcNAc (18B10.C7) (Thermo Fisher Scientific, MA1-038); FLAG (Sigma-Aldrich, F3165); HA (BioLegend, MMS-101P, clone 16B12); HA (Cell Signaling Technology, 3724); and Myc (Santa Cruz Biotechnology, sc-40).

Techniques: Labeling, Immunostaining, Western Blot, Whisker Assay, Transfection, Construct, Injection, Liquid Chromatography with Mass Spectroscopy

Journal: Nature chemical biology

Article Title: Direct stimulation of de novo nucleotide synthesis by O-GlcNAcylation.

doi: 10.1038/s41589-023-01354-x

Figure Lengend Snippet: Fig. 6 | R196W decreases PRPS1 O-GlcNAcylation and activity. a,b, HEK293T cells were transfected by indicated plasmids. Interactions between OGT and WT/mutant FLAG-PRPS1 (L152P, R196W and Q133P) were analyzed by co- immunoprecipitation (IP). PRPS1 O-GlcNAcylation in each group was examined (a) (n = 3). PRPS1 enzymatic activity from a is shown (b) (n = 3). c,d, Myc-OGT was transfected into WT or R196W mutant HEK293T cells, and PRPS1 O-GlcNAcylation level (c) and enzymatic activity (d) were examined (n = 3). e, PRPS1 R196W mutation inheritance and family phenotype and conservation of PRPS1 R196 residue across species. The stillbirth (SB) statuses with the gestational week are indicated in the pedigree chart. f, WT or PRPS1 R196W mutant from HEK293T cells was eluted in fractions on the size-exclusion column in the same settings. Indicated fraction samples were used for immunoblotting (n = 3). g, WT or PRPS1 R196W plasmids were replenished into PRPS1 knockout H1299 cells. EdU incorporation assays were performed as in

Article Snippet: The following primary antibodies were used for immunoblotting: PRPS1 (Proteintech, 15549-1-AP); phospho-PRPS1 (S180) (Thermo Fisher Scientific, PA5-106230); PRPS2 (Novus, NBP1-31435); OGT (Cell Signaling Technology, 24083); OGA (Proteintech, 14711-1-AP); AMPKα (Cell Signaling Technology, 2532); phospho-AMPKα (Thr172) (Cell Signaling Technology, 2535); ACC (Cell Signaling Technology, 3662); phospho-ACC (S79) (Cell Signaling Technology, 3661); β-tubulin (Proteintech, 66240-1-Ig); β-actin (Proteintech, 60008-1-Ig); γ-H2AX (S139) (Cell Signaling Technology, 9718); histone H3 (Millipore, 06-755); O-GlcNAc (RL2) (Abcam, ab2739); O-GlcNAc (CTD110.6) (Cell Signaling Technology, 9875); O-GlcNAc (18B10.C7) (Thermo Fisher Scientific, MA1-038); FLAG (Sigma-Aldrich, F3165); HA (BioLegend, MMS-101P, clone 16B12); HA (Cell Signaling Technology, 3724); and Myc (Santa Cruz Biotechnology, sc-40).

Techniques: Activity Assay, Transfection, Mutagenesis, Immunoprecipitation, Residue, Western Blot, Knock-Out

Journal: STAR Protocols

Article Title: An efficient and versatile CRISPR-Cas9 system for genetic manipulation of multi-drug resistant Klebsiella pneumoniae

doi: 10.1016/j.xpro.2021.100373

Figure Lengend Snippet:

Article Snippet: K. pneumoniae , ATCC , ATCC# BAA-1705D-5.

Techniques: Recombinant, Selection, Gel Extraction, Purification, Virus, Software, Polymer, Cell Culture, Sequencing